Injectable Nano Whitlockite Incorporated Chitosan Hydrogel for

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Injectable Nano Whitlockite Incorporated Chitosan Hydrogel for Effective Hemostasis Nivedhitha Sundaram Muthiah Pillai, Kalyani Eswar, Sivashanmugam Amirthalingam, Ullas Mony, Praveen Kerala Varma, and Rangasamy Jayakumar ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00710 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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ACS Applied Bio Materials

Injectable Nano Whitlockite Incorporated Chitosan Hydrogel for Effective Hemostasis Nivedhitha Sundaram Muthiah Pillai1, Kalyani Eswar1, Sivashanmugam Amirthalingam1, Ullas Mony1, Praveen Kerala Varma2 & Rangasamy Jayakumar1* 1Center

for Nanosciences and Molecular Medicine, 2Department of Cardio Vascular

and Thoracic Surgery, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, India

---------------------------------------------------------------------------*Corresponding Author. Rangasamy Jayakumar E-mail: [email protected] & [email protected] Tel: +91-484-280-1234 1 ACS Paragon Plus Environment

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ABSTRACT Uncontrolled bleeding can lead to many complications that might cause multi organ failures and even death. Of all the hemostatic agents used, chitosan has been reported to show better hemostatic potential. It acts through one mechanism involved in hemostasis that is plug formation by adhering to the injured site. Hence our focus is to enhance the hemostatic potential of chitosan (Ch) hydrogel by incorporating nano whitlockite (nWH: Ca18Mg2(HPO4)2(PO4)12) that would release Ca2+, Mg2+ and PO43- ions which would simultaneously initiate the coagulation cascade. Ch-nWH composite hydrogel can act simultaneously on different mechanisms involved in hemostasis and bring about rapid bleeding control. The nWH

particles

were

synthesized

using

precipitation

technique

and

were

characterized. Particle size of nWH was found to be 75 ± 5 nm. Composite hydrogel was characterized using FTIR and XRD to confirm the presence of different constituents of the hydrogel. Rheological studies showed the shear-thinning property and increased elastic modulus of the composite hydrogel compared to Ch hydrogel. 2%Ch-4%nWH hydrogel was observed to be cytocompatible with Human Umbilical Vein Endothelial Cells (HUVEC). In the in vitro blood clotting analysis using citrated human whole blood, 2%Ch-4%nWH hydrogel showed rapid blood clot formation compared to control 2%Ch hydrogel. Further in vivo experiments performed on liver and femoral artery injuries created on Sprague Dawley (S.D) rat model reveals that 2%Ch-4%nWH hydrogel promoted rapid bleeding control and less volume of blood loss compared to Ch hydrogel. These in vitro and in vivo results showed that incorporation of nWH has enhanced the hemostatic potential of Ch hydrogel. Therefore, the synthesized 2%Ch-4%nWH hydrogel may be a promising system that could bring about rapid hemostasis during life threatening bleeding. 2 ACS Paragon Plus Environment

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KEYWORDS:

Injectable

hydrogel,

Chitosan,

Whitlockite

nanoparticles,

Hemostatic agent, Hemostasis.

1. INTRODUCTION Body’s native hemostasis mechanism would not be able to effectively control bleeding during trauma or during certain surgical procedures where there is a severe blood loss.1 Hemostatic agent as an external agent can help to achieve hemostasis when normal coagulation pathway doesn’t function properly. Commonly used commercially

available

topical

hemostatic

agents

are

fibrin

glue,

thrombin/recombinant thrombin spray, gelatin sponge, chitosan sponges and dressings, collagen, oxidized cellulose, zeolite powder and alginate based agents.2 Chitosan (Ch) has received FDA approval to be used as a hemostatic agent and the commercially available Ch based hemostatic agents are Hemcon, Chitoflex, Celox-A and Syvek Excel.3 Main mechanism by which Ch based hemostatic agent act is by adhering and physically sealing the bleeding wound by forming a plug.4 This is mainly by the electrostatic interaction between positive charge of protonated amine group of Ch and negative charge of erythrocytes cell membranes.5 But the hemostatic efficiency of Ch has not yet met the need for severe bleeding wounds and it also requires compression at the injury site to bring about effective hemostasis.6,7 Hemostatic potential of Ch based hemostatic agents can be improved by incorporation of inorganic materials.8,9 Whitlockite (WH: Ca18Mg2(HPO4)2(PO4)12) is one of the component that constitutes approximately upto 20 wt% of the inorganic phase of human bone. WH consists of Ca2+, Mg2+ and PO43- ions that are potent activators of different coagulation factors involved in coagulation cascade.10 The Ca2+ ion play a major role in activating coagulation factors.11,12 Similarly Mg2+ ions is 3 ACS Paragon Plus Environment


also involved in blood coagulation cascade, it helps in stabilizing the native conformation of coagulation factor IX.13,14 The PO43- is involved in platelet activation and aggregation.15,16,17 WH in nano size (nWH) would have high affinity towards plasma proteins such as fibrinogen due to its high surface area to volume and would help in fibrin clot formation.18,19 Therefore, our focus is to develop a composite hydrogel that would seal the injured site by forming a plug by electrostatic interaction between positive charge of protonated amine group of Ch and negative charge of erythrocytes cell membranes and at the same time activate coagulation cascade by Ca2+, Mg2+ and PO43- ions released from nWH to bring about rapid and effective hemostasis. In this study, we have developed an injectable Ch-nWH composite hydrogel and evaluated its injectablility, cytocompatibility, blood clotting time in vitro and hemostatic potential in vivo in liver and femoral artery bleeding conditions created in rat model.

2. EXPERIMENTAL SECTION 2.1. Materials. Ch (Avg MW:100-150 kDa; DD:85%) was obtained from Koyo, Japan. Calcium hydroxide, magnesium hydroxide, hydrogen peroxide, glacial acetic acid, milliQ water, sodium hydroxide, triton-X 100 were obtained from Spectrochem Pvt. Ltd., India. IMDM and large-vessel endothelial supplement (LVES) were purchased from Invitrogen, India. 0.25% trypsin-EDTA and FBS were also obtained from Invitrogen, India. Saline solution and Sodium citrated blood collection tubes were purchased from Infutec Healthcare Ltd., India and BD, USA respectively. Floseal hemostatic matrix was purchased from Baxter, USA. 2.2. Method. 2.2.1. Preparation of Whitlockite Nanoparticles (nWH). The nWH particles was prepared by following previously published protocol.20 Nanoparticle

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preparation is by precipitation technique that involves addition of calcium hydroxide (0.37M) and magnesium hydroxide (0.13M) to water under vigorous stirring maintained at 80 °C for 1 h, followed by dropwise addition of phosphoric acid (0.5 M) to the mixture. After 18 h of aging, the precipitate was centrifuged (at 9500 rpm for 20 mins in HERMLE Z 326 K centrifuge) and washed thrice with double distilled water; finally freeze dried to get nWH particles (Figure 1A).

Figure 1. Schematic representation of steps involved in synthesis of (A) nWH powder, (B) 2%Ch hydrogel and (C) 2%Ch-4%nWH composite hydrogel. 2.2.2. Preparation of 2%Ch-4%nWH composite hydrogel. Ch powder (2g) was dissolved in 1% acetic acid solution (100 mL) and regenerated by dropwise addition of 1% NaOH. Regenerated 2%Ch hydrogel was obtained by centrifugation at 9500 rpm for 20 mins in HERMLE Z326K centrifuge (with 220.78 rotor). Ch hydrogel was 5 ACS Paragon Plus Environment


then washed (by centrifugation) three times using double distilled water to remove excess NaOH21 (Figure 1B). 2%Ch-4%nWH composite hydrogel was prepared by the addition of nWH (4w/w %) to Ch hydrogel under uniform mixing (Figure 1C). 2.2.3. Physiochemical Characterization of prepared nWH and 2%Ch-4%nWH composite hydrogel. Surface morphology and size of the prepared nWH particles were observed using Transmission Electron Microscopy (TEM, FEI Tecnai F20). The nWH particle suspension (5 µL) was dropped on copper grid, air dried and then analyzed. 2%Ch and 2%Ch-4%nWH hydrogel systems were analyzed using Scanning Electron microscope (SEM). After complete air drying of the hydrogel systems, they were placed on aluminium stub, sputter coated with gold (10 mA for 90 s; JEOL JFC-1600) and imaged at 15 kV. X-ray diffraction (XRD) analysis was done to confirm the presence of nWH in the 2%Ch-4%nWH composite hydrogel system. The characteristic XRD peaks of nWH were also compared to WH JCPDS (70-2064) database. FTIR (Shimadzu IR Affinity-1S) analysis was carried out at frequency range of 4000-500 cm-1 to obtain the characteristic spectra of lyophilized 2%Ch and 2%Ch-4%nWH hydrogel systems. Release of ions from nWH was also analyzed. 1wt% of nWH was incubated in double distilled water for 1, 24 and 48 h at room temperature. To quantify the amount of Ca, Mg and P ions release from nWH, after the specific time points the incubated solution was centrifuged at 4000 rpm for 30 mins and the supernatant was analyzed using ICP-AES; OPTIMA 8300 (Perkin-Elmer, USA). 2.2.4. Rheological Properties of Ch-nWH composite gel system. Rheological properties of the prepared hydrogel systems were analyzed using a rheometer (Malvern Kinexus Pro, UK) that is equipped with parallel plate geometry (20 mm diameter; 0.5 mm gap). The viscoelastic behavior of 2%Ch hydrogel and 2%Ch-


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4%nWH composite hydrogel systems were analyzed by performing amplitude sweep analysis, frequency sweep analysis and temperature stability studies by following previously established protocol.22 Briefly, Linear viscoelastic (LVE) region was determined and rheological studies were carried out in this region at 37°C (mimic physiological temperature). These studies were performed to evaluate the strength and stability of 2%Ch hydrogel and 2%Ch-4%nWH composite hydrogel systems. Frequency sweep was carried out from 101 to 10-1 Hz. For temperature stability analysis, temperature was ramped up from 25°C to 50°C at a ramping rate of 5°C/min. For flow curve analysis, shear rate was step wise increased from 0.1 to 100 s-1. Injectablility test was carried out to study the injectability property and smoothness of the developed 2%Ch hydrogel and 2%Ch-4%nWH composite hydrogel systems. Injectability property of the hydrogel systems were observed visually by loading hydrogel system (2g) onto 1 mL syringe and injecting it manually. Inversion test was performed to know if the developed hydrogel systems showed time-dependent shear-thinning property. This study was performed as per previously published protocol.23 Briefly, 2g hydrogel was taken in a flat-bottomed 10 mL storage vial (Tarsons, India) and was placed in an inverted position on flat surface. The inverted storage vials were undisturbed throughout the study. At desired time points (0th h and after 72 h) photographs of the inverted vials were taken. 2.2.5. In vitro cytocompatibility of nWH paricles and 2%Ch-4%nWH composite hydrogel system. Alamar Blue assay was performed to quantify live cells in contact with different percentages of nWH particles and also 2%Ch-4%nWH composite hydrogel system. HUVEC was isolated from umbilical cord according to reported protocol24 and was then cultured in IMDM containing AA, FBS and LVES. HUVEC



was seeded at a seeding density of 5 X 104 cells per well in a 24 well plate to which different percentages of nWH (0.5,1,2,3,4,5,6 & 7%), 2% Ch (100 mg) and 2% Ch4% nWH (100 mg) hydrogel systems were added. After incubation in CO2 incubator (at 37 °C, 5% CO2) for 48h, assay was performed. After specified time point, culture medium with nWH and hydrogel system was completely removed by washing with PBS. Upon complete removal of nWH and hydrogel system from each well, 500 μL of basal IMDM media containing 10% Alamar Blue reagent was added. After 6h of incubation of the well plate in cell culture incubator, optical density (OD) value of the reagent was obtained at 570 and 600 nm with the help of microplate spectrophotometer instrument (Biotek PowerWave XS, USA).25 HUVEC cultured for the specified time points without the addition of hydrogel served as control. 2.2.6 In Vitro Blood Clotting Time Analysis. In vitro blood clotting potential of 2%Ch and 2%Ch with increasing percentage of nWH (after 48 h incubation) hydrogel systems was evaluated by following previous published protocol with slight modification.26 100 mg of hydrogel systems were spread evenly to completely cover the bottom surface of wells of a 48 well plate. To these wells, 300 μL recalcified human whole blood solution (50 μL of 25 mM CaCl2 added to 250 μL calcified blood) was added. At specific time points, the wells were washed using saline (0.9% NaCl) to completely remove the unclotted blood components. Time at which there was formation of uniform and stable clot in the wells after washing gives the blood clotting time. 2.2.7. Hemolysis Assay. Fresh human whole blood was collected in 3.8% sodium citrated BD vacutainer tube. Blood collected was centrifuged and RBCs was separated as per previous established protocol.27 1 mL of concentrated RBCs was diluted with 9 mL saline. To evaluate the hemolytic activity of the developed hydrogel


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systems, 100 mg of 2% Ch and 2% Ch-4% nWH (after 48 h incubation) hydrogel was taken separately in vials. 0.1% Triton-X (100 μL) and saline (100 μL) were also taken in different vials, this served as positive and negative controls. To these vials, diluted RBCs (500 μL) was added and the vials were kept at 37 °C for 1h. After 1 h incubation samples were centrifuged (3500 rpm; 10 min) to obtain supernatant. Collected supernatant was read at 540 nm using a well plate reader (Biotek, power wave XS, US) to obtain its OD value. Hemolysis (%) was then calculated from the OD values using the formula mentioned below27:

2.2.8. In vivo hemostatic potential of 2% Ch-4% nWH composite hydrogel system in Liver Injury (oozing bleeding model) and Femoral Artery Injury (pressured bleeding model) created in Rat. Sprague Dawley rats (male, 250-300 g, 8 week old) were used for this study. Animal studies were performed following protocols approved by institution animal ethical committee. Surgical procedures for this study were done following the previous established protocol.26 For creation of liver injury, rats were anesthetized; abdomen incision was made; liver was exposed and fluid around the liver was completely removed to avoid inaccuracies in evaluating the mass of blood loss. 5 mm biopsy punch was used to create injury in the liver. For creation of femoral artery injury, rats were anesthetized; after exposing femoral artery, an injury was created by puncturing the artery using a 24 gauge needle. In both the studies; immediately after the injury was created, 200 mg of hydrogel was applied at the bleeding site. Time for hemostasis and mass of blood loss was then evaluated. Upon completion of necessary evaluation all animals used in this study were sacrificed. For the evaluation of mass of blood loss weighed gauze pieces were used in the study. Four rats per group was used in this study and the groups tested 9 ACS Paragon Plus Environment


are sham control, 2%Ch hydrogel, 2%Ch-4%nWH composite hydrogel (after 48 h incubation) and Floseal a commercially available injectable hemostatic agent. 2.2.9. Statistical Analysis. Results of all the experiments are denoted as mean ± SD with n > 3. Difference between groups was statistically analysed using ANOVA and Tukey’s test. P value less than 0.05 was taken as statistically significant.

3. RESULTS AND DISCUSSION 3.1. Preparation and physiochemical characterization of composite hydrogel. Ch-nWH composite hydrogel was prepared by first preparing 2%Ch hydrogel by simple regeneration chemistry by changing pH and then uniformly distributing 4%nWH in the hydrogel. SEM and TEM images of the prepared nWH particles showed its uniform morphology, rhombohedral shape and particle size of 75 ± 5 nm (Figure 2A&B). SEM image of the prepared hydrogel systems showed the smooth morphology of 2%Ch hydrogel and homogeneous distribution of nWH in the composite hydrogel was observed (Figure 2C&D).


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Figure 2. (A) SEM and (B) TEM images of the prepared nWH particles, SEM image of the developed (C) 2%Ch and (D) 2%Ch-4%nWH hydrogel systems. Presence of nWH in the developed composite hydrogel was further confirmed using FTIR. FTIR result of the developed composite hydrogel (Figure 3A) showed the characteristics peaks of nWH20 at 559, 605 and 918 cm-1 (−PO4 stretching) and characteristics peaks of Ch21 at 1070 (-C-O stretch), 1425, 1566 (-N-H stretch) and 2920 cm-1 (-C-H stretch) indicating the presence of nWH in the composite hydrogel. The prepared nWH particles were further characterized using XRD analysis. XRD peaks obtained for nWH particles matched with the XRD peaks of WH from JCPDS data (70-2064).28 In the XRD spectra of composite hydrogel the characteristic peak of nWH at 2θ of 31.2, 34.5 and 47.2° which correspond to the plane (0 2 10), (2 2 0) and (4 0 10) respectively was observed (Figure 3B). This further confirmed the presence of nWH in the composite hydrogel.



Figure 3. (A) FTIR spectra, (B) XRD peaks of the developed nWH particles, 2%Ch hydrogel and 2%Ch-4%nWH composite hydrogel and (C) Amount of Ca, Mg and P ions release from nWH. Ca, Mg and P ions released after 1, 24 and 48 h from 1%nWH dispersed in double distilled water at neutral pH were measured using ICP-AES. At all-time points, release of Ca ions was more than Mg and P ions. By 48 h, nearly 12.6 ± 0.6 mg/L of Ca ions; 2.2 ± 0.1 mg/L of Mg ions and 6.1 ± 0.2 mg/L P ions were released from nWH (Figure 3C). These ions released from nWH might act simultaneously to help in achieving effective hemostasis. 3.2. Rheological studies. Amplitude sweep analysis was carried out to find the linear viscoelastic (LVE) region of the hydrogel. Further rheological experiments were carried out in the LVE region. The storage modulus of 2%Ch hydrogel was found to be 10.46±1.5 kPa, further on addition of WH nanoparticles, the storage modulus had increased to 31.27±0.77 kPa. The storage modulus value (G’) of the hydrogel systems was found to be greater than loss modulus (G’’) indicating its solid like nature22. Phase angle of the developed hydrogels were also found be less than 45° hence proving its solid dominant property23. The phase angle was found to be 4.80±1.3° and 5.83±0.9° for 2%Ch-4%nWH and 2%Ch gel, respectively. The developed hydrogel systems was subjected to frequency sweep analysis (101 to 10-1 Hz) and from the results it was noted that the G’ value of the developed 2%Ch and 2%Ch-4%nWH hydrogel systems remained stable over the frequency range (Figure 4A). Temperature stability of the hydrogels was evaluated. Temperature was ramped up from 25 to 50°C and the rheological parameters were monitored. During the study range, the storage modulus didn’t change (Figure 4B). This indicates that the prepared hydrogel wouldn’t be affected post injection into the body20. Flow curve


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analysis was performed so as to study the shear thinning behaviour of the prepared hydrogels. The experiment was carried out from 0.1 s-1 to 100 s-1. The viscosity of the Ch hydrogel was reduced from 5739 Pa.s to 2.078 Pa.s, whereas for the ChnWH hydrogel, the viscosity was reduced from 31250 Pa.s to 2.258 Pa.s after application of 1000 fold (0.1 to 100 s-1) increase in shear rate (Figure 4C). This study showed that application of high rate, the viscosity of the hydrogel drops down to 2 Pa.s, which showed that the prepared hydrogel was injectable shear thinning hydrogel. To observe the phenomena visually, hydrogel was loaded into 1ml syringe and it was injected. Both the hydrogel showed smooth and continuous flow while injection (Figure 4D). Further, hydrogel was injected into the storage vial and it was kept undisturbed. Hydrogel didn’t exhibit flow due to gravitational force (Figure 4E). These results show that the developed hydrogel could be easily injected into the bleeding site.



Figure 4. Rheological properties of the developed 2%Ch and 2%Ch-4%nWH hydrogel systems, (A) Frequency sweep analysis of 2%Ch-4%nWH hydrogel and 2%Ch hydrogel representing G’ and δ, (B) Temperature stability study from 25° to 50°C, (C) Flow behaviour with respect to shear rate; ramp down and ramp up of 2%Ch-4%nWH and 2%Ch hydrogel systems, (D) Injectablility study and (E) Inversion test of the developed hydrogel systems. The dotted lines in (A) and (B) represents phase angle baseline to determine whether the tested material solid dominant or liquid dominant; values below baseline is solid-dominant and values above baseline is liquid dominant. 3.3. In vitro cytocompatibility of composite hydrogel system. Cytocompatibility of different concentrations of nWH and developed 2%Ch-4%nWH hydrogel system in the presence of HUVEC was evaluated using alamar blue assay. Results show that upon increasing the concentration of nWH (>4%) showed slight decrease in viability of HUVEC. Therefore 4%nWH was chosen for further studies (Figure 5A). Higher concentration of nanoparticles might have induced stress to cells this would have caused the decrease in viability. Cytocompatibility of the developed hydrogel system was also performed and its results show no statistical significant difference between percentage viability of cells alone and cells in contact with the hydrogel systems (2%Ch and 2%Ch-4%nWH) (Figure 5B).


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Figure 5. Percentage cell viability of (A) Different concentrations of nWH and (B) developed 2%Ch hydrogel and 2%Ch-4%nWH composite hydrogel systems in contact with HUVEC. 3.4. In vitro clotting ability of developed composite hydrogel system. In vitro blood clotting potential of the developed 2%Ch hydrogel and 2%Ch-4%nWH composite hydrogel was studied by evaluating the blood clotting time when the developed hydrogel systems come in contact with citrated human whole blood. Blood clotting time of whole blood alone (without hydrogel) was found to be 7.2 ± 0.5 min which is similar to normal human whole blood clotting time reported in literature.29 2%Ch hydrogel when in contact with whole blood, the amine groups in Ch hydrogel would have stimulated blood clot formation5 but a stable clot was formed only by 5.5 ± 1 min (Figure 6A). Only upon incorporation of different percentages of nWH particles in to 2%Ch hydrogel and incubation of composite hydrogel for release of ions from nWH the clotting time decreased. 2%Ch-4%nWH composite hydrogel after 48 h incubation with 4% nWH particles showed less blood clotting time of 3.5 ± 1.2 min (Figure 6B). Incubation of composite hydrogel for 48 h showed significant reduction in the blood clotting time. Incubation of 2%Ch-4%nWH composite hydrogel for 24 h didn’t show significant reduction in the blood clotting time compared to 2%Ch hydrogel. Incorporation of nWH particles into composite hydrogel and incubation for 48 h has reduced the blood clotting time due to the simultaneous action of Ca2+, Mg2+ and PO43- ions released from nWH along with amine groups of Ch hydrogel. 3.6. Hemolysis assay. Hemocompatibility of the developed 2%Ch hydrogel and 2%Ch-4%nWH composite hydrogel systems was evaluated by performing the hemolysis assay. Photographic image of vial containing saline, 2%Ch gel, 2%Ch-



4%nWH gel and Triton X treated with diluted RBCs after centrifugation was obtained (Figure 6C). From the photographic image it was observed that the supernatant of the samples was clear and was similar to saline treated diluted RBCs. This reveals the non-hemolytic property of the samples. Further, percentage hemolysis of the hydrogel systems was found to be less than 5% which is an allowable limit for biomaterials (Figure 6D). Therefore, Ch hydrogel and composite hydrogel were found to be hemocompatible.

Figure 6. Effect of 2%Ch hydrogel and 2% Ch-4%nWH composite hydrogel when treated with human blood, (A) Photographic image of blood clot formed with respect to time, (B) Blood clotting time of the developed hydrogel systems, (C) Photographic image showing hemolysis and (D) Percentage hemolysis of the developed hydrogel systems. (** represents P< 0.001) (*** represents P< 0.001) 3.7. In vivo hemostatic potential of 2%Ch-4%nWH composite hydrogel. In vivo hemostatic potential of 2%Ch-4%nWH composite hydrogel was compared with 2%Ch hydrogel and Floseal (commercially available injectable hemostaitc agent) in 16 ACS Paragon Plus Environment

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lethal bleeding conditions like liver (Figure 7A) and femoral artery injury (Figure 8A) models.

Figure 7. (A) Surgical procedure for creation of Liver injury in rat, (B) Time taken for hemostasis and (C) Mass of blood loss after application of 2%Ch hydrogel, 2%Ch4%nWH hydrogel and Floseal. (** indicates P< 0.01) 17 ACS Paragon Plus Environment


Liver, a highly vascularized organ was injured to study blood ooze condition. Liver injury was created and 2%Ch hydrogel (200mg), 2%Ch-4%nWH composite hydrogel (200 mg) and Floseal (200mg) were applied individually on the bleeding site. Bleeding caused due to injury in sham control (injury untreated with hydrogel) took 147 ± 7 sec to achieve hemostasis. When 2%Ch hydrogel was treated on the injured site, time for hemostasis obtained was 93 ± 3 sec and when Floseal the commercial hemostatic agent was applied, time taken for hemostasis was 88 ± 4 sec. 2%Ch-4%nWH composite hydrogel when applied to injured site was treated the time to achieve hemostasis decreased and was found to be 62 ± 3 sec (Figure 7B). Mass of blood loss was less when 2%Ch-4%nWH composite hydrogel (478 ± 8 mg) was applied in comparison with 2%Ch hydrogel (552 ± 9 mg), Floseal (514 ± 16 mg) and sham control (901 ± 15 mg) (Figure 7C).


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Figure 8. (A) Surgical procedure for the creation of Femoral artery injury in rat, (B) Time taken for hemostasis and (C) Mass of blood loss after application of 2%Ch hydrogel, 2%Ch-4%nWH hydrogel and Floseal. (** indicates P< 0.01; * indicates P

Injectable Nano Whitlockite Incorporated Chitosan Hydrogel for

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